2011 Annual Report 1a.Objectives (from AD-416) 1. Obtain complete annotated genome sequence of the zebra chip disease bacterium ‘Ca. Liberibacter psyllaurous/solanacearum’. 2. Identify the effectors and other pathogenesis-related genes of ‘Ca. Liberibacter solanacearum’. 3. Use the genome information to generate genetic markers, such as SSR, for detection and differentiations of the PY and ZC bacterial complex. 1b.Approach (from AD-416) 1. Comparative gene order mapping of Lps contigs using the genomes of three phylogenetically related organisms, Candidatus Liberibacter asiaticus, Sinorhizobium meliloti and Bartonella quintana, as potential scaffolds using Circos software (available online), design PCR primers, Amplified DNA will be then sequenced and used to close gaps of the chromosome. Meanwhile, construction of genomic libraries to close Lps genome gaps and to obtain other Lps sequence information. 2. Annotate genes, gene products and metabolic pathways to assess the nutritional supplements required for in vitro culture of Lps. Re-sequencing of low coverage genomic regions and putative pseudogenes. 3. Comparative genomics of Lps/Lso and Las, reconstruction of predicted ‘Ca. Liberibacter solanacearum’ metabolic pathways will be carried out using IdentiCS software (Sun and Zeng 2004). Annotated enzyme models will be used to map the ‘Ca. Liberibacter solanacearum’ data onto the KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathways (Kanehisa and Goto 2000; Kanehisa et al. 2006, 2008). A comparison between ‘Ca. Liberibacter solanacearum’ proteins and Ca. Liberibacter asiaticus proteins in the Transporter Classification database of the Saier Lab Bioinformatics Group (available online) will be made to classify and group transporter proteins in the newly annotated genome according to the widely accepted Transporter Classification system. 4. Generate genetic markers for detection and differentiation of PY and ZC strains of Lps/Lso. Archived DNA from PY and ZC affected potato plants will be used in this analysis as well as DNA from PY and ZC grafted plants. 3.Progress Report This project is related to inhouse objective 1: Characterize ecology, biology, epidemiology, genetics and host interactions of domestic, exotic, newly emergent and re-emerging pathogens. In collaboration with the USDA-ARS in Parlier, California, we have completed the genome sequence of ‘Candidatus Liberibacter solanacearum’ with ca.1.26Mb. Based on the genome sequence, which has been published in PLoS ONE 6(4): e19135.doi:10.1371/journal.pone.0019135. Thirty five pairs of PCR markers were designed and used to screen 17 potato samples consisting of 5 plants infected with psyllid yellows (PY), 3 plants infected with zebra chip (ZC), 3 plants affected by haywire (HW), 3 asymptomatic plants (NZC) and 3 plants affected with an unknown disease (UNK). The results suggest there is significant genetic variation between asymptomatic NZC but Lso positive plants and other types of potato samples tested and that two different genotypes may be involved in ZC (type 1 and type 2). Using two sets of primers and probes, we were able to differentiate these two genotypes by quantitative realtime polymerase chain reaction (qPCR). These results provide further evidence of the existence to two genotypes of Lso involved in zebra chip disease of potato. Progress was monitored via through direct involvement in lab and field activities, research meetings, telephone calls and email communications.